Loss of functional skeletal muscle due to congenital and acquired conditions, such as traumatic injury, tumor excision, etc., produces a physiological deficit for which there is still no effective clinical treatment. Tissue engineering of skeletal muscle in vitro for functional tissue replacement in vivo may provide a potential therapeutic solution to this unmet medical need. To this end, we will use our established approach in which primary muscle precursor cells (MPCs) are seeded onto collagen-based acellular tissue scaffolds. This approach will be combined with two enabling technologies to build functional muscle tissue that could be translated clinically;first, a cyclic strain protocol in a custom designed, computer-controlled bioreactor system, and second, utilization of a novel class of oxygen generating biomaterials. The first technology consists of cyclic mechanical strain in vitro leading to enhanced skeletal muscle contractility in vivo, while the second will evaluate the working hypothesis that incorporation of oxygen generating particles in the scaffold will permit prolonged muscle tissue survival until host vascularization is established in vivo. As such, this proposal is clearly consistent with PAR-06-504 "Enabling Technologies for Tissue Engineering and Regenerative Medicine (R01)". To achieve our long-term goal of increased bioengineered skeletal muscle tissue function and mass in vivo, we propose a step-wise increase in size and complexity of the bioengineered muscle tissue constructs;for time points ranging from 1-16 weeks post-implantation. We will monitor and assess the phenotypic and functional maturation of the tissue engineered constructs both in vitro and in vivo, using a multidisciplinary approach (on both engineered and native tissue from the same animal) ranging from the whole tissue to the single fiber, molecular and genetic levels. We will leverage the unique features of the latissimus dorsi functional replacement model to evaluate the ability of our enabling technologies to achieve bioengineered skeletal muscle that approximates native skeletal muscle. Specific Aim #1: To evaluate the impact of bioreactor preconditioning and particulate oxygen generators (POGs) on the formation and function of skeletal muscle implanted onto the latissimus dorsi (LD) in vivo (Years 1-2). Specific Aim #2: Application of bioengineered skeletal muscle for functional replacement in a small defect in a mouse latissimus dorsi model: "Proof of Concept" (Years 2-3). Specific Aim #3: Development of bioengineered skeletal muscle for functional replacement in a large defect in a mouse LD model: "Evaluation of clinical applicability" (Years 4-5). Public Health Relevance Statement (provided by applicant): Loss of functional skeletal muscle due to congenital and acquired conditions, such as traumatic injury, tumor excision, etc., produces a physiological deficit for which there is still no effective clinical treatment. Tissue engineering of skeletal muscle in vitro for functional tissue replacement in vivo may provide a potential therapeutic solution to this unmet medical need.